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      Comparison of Measurements of Autoantibodies to Glutamic Acid Decarboxylase and Islet Antigen-2 in Whole Blood Eluates from Dried Blood Spots Using the RSR-Enzyme Linked Immunosorbent Assay Kits and In-House Radioimmunoassays

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          Abstract

          To evaluate the performance of dried blood spots (DBSs) with subsequent analyses of glutamic acid decarboxylase (GADA) and islet antigen-2 (IA-2A) with the RSR-ELISAs, we selected 80 children newly diagnosed with type 1 diabetes and 120 healthy women. DBSs from patients and controls were used for RSR-ELISAs while patients samples were analysed also with in-house RIAs. The RSR-ELISA-GADA performed well with a specificity of 100%, albeit sensitivity (46%) was lower compared to in RIA (56%; P = .008). No prozone effect was observed after dilution of discrepant samples. RSR-ELISA-IA-2A achieved specificity of 69% and sensitivity was lower (59%) compared with RIA (66%; P < .001). Negative or low positive patients and control samples in the RSR-ELISA-IA-2A increased after dilution. Eluates from DBS can readily be used to analyse GADA with the RSR-ELISA, even if low levels of autoantibodies were not detected. Some factor could disturb RSR-ELISA-IA-2A analyses.

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          The cation efflux transporter ZnT8 (Slc30A8) is a major autoantigen in human type 1 diabetes.

          Janet Wenzlau, Kirstine Juhl, Liping Yu … (2007)
          Type 1 diabetes (T1D) results from progressive loss of pancreatic islet mass through autoimmunity targeted at a diverse, yet limited, series of molecules that are expressed in the pancreatic beta cell. Identification of these molecular targets provides insight into the pathogenic process, diagnostic assays, and potential therapeutic agents. Autoantigen candidates were identified from microarray expression profiling of human and rodent pancreas and islet cells and screened with radioimmunoprecipitation assays using new-onset T1D and prediabetic sera. A high-ranking candidate, the zinc transporter ZnT8 (Slc30A8), was targeted by autoantibodies in 60-80% of new-onset T1D compared with <2% of controls and <3% type 2 diabetic and in up to 30% of patients with other autoimmune disorders with a T1D association. ZnT8 antibodies (ZnTA) were found in 26% of T1D subjects classified as autoantibody-negative on the basis of existing markers [glutamate decarboxylase (GADA), protein tyrosine phosphatase IA2 (IA2A), antibodies to insulin (IAA), and islet cytoplasmic autoantibodies (ICA)]. Individuals followed from birth to T1D showed ZnT8A as early as 2 years of age and increasing levels and prevalence persisting to disease onset. ZnT8A generally emerged later than GADA and IAA in prediabetes, although not in a strict order. The combined measurement of ZnT8A, GADA, IA2A, and IAA raised autoimmunity detection rates to 98% at disease onset, a level that approaches that needed to detect prediabetes in a general pediatric population. The combination of bioinformatics and molecular engineering used here will potentially generate other diabetes autoimmunity markers and is also broadly applicable to other autoimmune disorders.
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            Diabetes Antibody Standardization Program: evaluation of assays for autoantibodies to glutamic acid decarboxylase and islet antigen-2.

            Paul Schlosser, Jonathan Mueller, … (2008)
            Islet autoantibodies are important in diabetes classification and risk assessment, and as endpoints in observational studies. The Diabetes Autoantibody Standardization Program (DASP) aims to improve and standardise measurement of autoantibodies associated with type 1 diabetes. We report results for glutamic acid decarboxylase autoantibodies (GADA) and islet antigen-2 autoantibodies (IA-2A) from three DASP workshops (2002--2005). Up to 60 laboratories in 18 countries participated in each workshop. Participants received coded serum aliquots from 50 patients with newly diagnosed type 1 diabetes (median age 18 years, range 9-35 years) and 100 blood donor controls. Results were analysed using receiver operator characteristic (ROC) curves with sensitivity adjusted to 95% specificity in workshop controls. GADA assays performed well in all three workshops (median area under the ROC curve [AUC] 0.94; interquartile range 0.91-0.95) and performance was similar to DASP 2000. Performance of IA-2A assays improved over the workshop programme. Median AUC was 0.81 (interquartile range 0.79-0.83) in DASP 2002, 0.82 (interquartile range 0.78-0.84) in 2003, and 0.85 (interquartile range 0.82-0.87) in 2005 (p < 0.0001). Performance of GADA ELISA improved between 2002 and 2005, and, in DASP 2005, achieved higher median AUC and adjusted sensitivity than RIA. IA-2A ELISA improved and, in DASP 2005, achieved AUCs equivalent to in-house RIA. Assays using IA-2ic or full length IA-2 clones were more sensitive than those using IA-2bdc, with higher AUC (p = 0.004). GADA and IA-2A assays perform well in discriminating health and disease. The workshop format highlights systematic differences related to assay method and allows full evaluation of novel methods. The programme of autoantibody workshops in type 1 diabetes provides a model for other autoimmune diseases.
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              Diabetes Antibody Standardization Program: first assay proficiency evaluation.

              Polly J. Bingley, Jonathan Mueller, Ezio Bonifacio (2003)
              The aims of the first proficiency evaluation of the Diabetes Antibody Standardization Program (DASP) were to assess general implementation of assay methods and to evaluate the new World Health Organization (WHO) reference reagent for autoantibodies to GAD and IA-2. Forty-six laboratories in 13 countries received coded sera from 50 patients with newly diagnosed type 1 diabetes and 50 blood donor control subjects, together with the WHO reference reagent and diluent serum. Results were analyzed using receiver operator characteristic (ROC) curves. Sensitivity was adjusted to 90% specificity in workshop controls. The median adjusted sensitivity for GADA (45 laboratories) was 84% (range 62-96%), for IA-2A (43 laboratories) was 58% (50-74%), and for insulin autoantibody (IAA; 23 laboratories) was 36% (13-66%). ROC curve analysis showed all GADA and IA-2A assays, and 18/23 IAA assays found significant differences between patients and control subjects. There was good concordance between laboratories in ranking of samples by GADA and IA-2A levels or if results were expressed in relation to the WHO reference reagent. Assays that achieved the highest sensitivity for IAA were also concordant in ranking samples, but overall concordance for IAA was poor. Differences in assay protocols between laboratories must be addressed so that all centers and kit manufacturers can perform to the same high standard.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Exp Diabetes Res
                Journal ID (publisher-id): EDR
                Title: Experimental Diabetes Research
                Publisher: Hindawi Publishing Corporation
                ISSN (Print): 1687-5214
                ISSN (Electronic): 1687-5303
                Publication date (Print): 2010
                Publication date (Electronic): 3 June 2010
                Volume: 2010
                Electronic Location Identifier: 173652
                Affiliations
                1Unit for Diabetes and Celiac Disease, Department of Clinical Sciences, Lund University, Wallenberg Laboratory, Entrance 46, University Hospital MAS, 205 02 Malmö, Sweden
                2Department of Clinical Chemistry, University Hospital MAS, 205 02 Malmö, Sweden
                3Unit for Diabetes and Celiac Disease, Department of Clinical Sciences, Lund University, Clinical Research Center (CRC), University Hospital MAS, 205 02 Malmö, Sweden
                Author notes

                Academic Editor: Rodica Pop-Busui

                Article
                DOI: 10.1155/2010/173652
                PMC ID: 2896843
                PubMed ID: 20613950
                SO-VID: c70c3a32-3f41-416f-8d39-52c0b02f77f6
                Copyright © Copyright © 2010 Anders Persson et al.
                License:

                This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 26 June 2009
                Date revision received : 7 September 2009
                Date accepted : 24 March 2010
                Categories
                Subject: Methodology Report

                ScienceOpen disciplines: Endocrinology & Diabetes
                Data availability:
                ScienceOpen disciplines: Endocrinology & Diabetes

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